Competitive binding assay immunoassay for platelet antigens in whole blood

ABSTRACT

A competitive binding assay kit and process for the immunological detection of a blood antigen of specified phenotype (for example phenotypes of human platelet antigen HPA) in a sample of whole blood. The kit contains a substrate having purified blood antigen of specified phenotype immobilized thereon, antibody specific to said blood antigen and means for enabling detection of antibody bound to said blood antigen. The process comprises mixing a sample of whole blood with a predetermined amount of antibody, so as to bind the antibody to any antigen present in the sample of whole blood and bringing the mixture into contact with the immobilized antigen so as to bind any remaining unbound antibody to the immobilized antigen, and determining the amount of immobilized complex and consequently the specified blood antigen phenotype.

FIELD OF THE INVENTION

The present invention relates to a competitive binding assay kit andprocess for the immunological detection of a presence of a blood antigenof specified phenotype (for example phenotypes of HPA) in a sample ofwhole blood.

BACKGROUND OF THE INVENTION

Human Platelet Antigen 1 (HPA1), also known as PLA1, is a polymorphicdeterminant on the glycoprotein (GP) IIb/IIIa complex, that resides atthe N-terminal region of GPIIIa. The great majority of the Caucasianpopulation is homozygous or heterozygous for HPA1a (about 72% and 26%respectively) (1). However, 2-3% of the population is homozygous forHPA1b (PLA1 negative) putting them at risk from developing antibodies toHPA1a either during pregnancy or following transfusion with bloodcontaining HPA1a platelets. For example, HPA1b pregnant women carryingHPA1a positive babies may produce anti-HPA1a antibodies which could leadto fetal or neonatal alloimmune thrombocytopenia (FAITP or NAITP)respectively, with 50% of the cases occurring at first pregnancy. Oneoutcome of FAITP/NAITP, which has a frequency of 1 in 1000-2000 births,is intracranial hemorrhage with 6.5-10% mortality and 20% morbidity(neurological sequelae) (2).

Moreover, HPA1b individuals (mostly elderly multi-parous women)transfused with blood containing HPA1a platelets may developpost-transfusion purpura (PTP) which is characterized with severethrombocytopenia, 5-10 days following the transfusion. Although not ascommon as NAITP (only about 200 cases reported world-wide), PTP isnevertheless associated with 10-20% morbidity/mortality (3).

The treatment and management of FAITP/NAITP and PTP relies basically onseveral infusions of intra-venous IgG with or without transfusion withHPA1b platelets. In view of the serious or fatal outcomes of FAITP/NAITPand PTP and their increasingly effective treatment, antenatal screeningfor HPA1b is becoming widely accepted as an important prophylacticmeasure (4,5). For such wide-scale screening, established assays such asmonoclonal antibody immobilization of platelet antigens (MAIPA),platelet suspension immunofluorescence test, or flow cytometry are notsuitable due to their technical or financial burdens (6-8). Recently,Metcalfe et al reported a simplified method for large-scale HPA1aphenotyping for antenatal screening which was adapted from MAIPA (9).However, the Metcalfe et al assay relies on the extraction ofglycoprotein GPIIb/IIIa antibody complex from platelets pre-incubatedwith the antibody. The assay is also considerably time consumingrequiring a number of incubation and centrifugation steps.

EP 0028133 describes a method of detecting and quantitating haptens andantigens. A competitve binding assay is described in which a sample anda reagent containing an immunoreactive antibody are in a liquid phase,the sample and reagent either being mixed together or reacted beforecontacting a solid phase onto which a known concentration of a substanceselected from haptens, antigens and/or mixtures thereof, which are alsoreactive with said antibody, is bound.

There is thus a requirement for an immunoassay which does not require asample of whole blood to be substantially purified.

It is an object of the present invention to obviate and/or mitigate someof the above disadvantages.

Generally speaking the present invention provides a simple andpotentially automatable competitive binding assay and assay kit fordetermining the presence in vitro of a specified blood antigen (forinstance HPA1a) in a sample of whole blood. It is generally based on thesurprising feature that the assay is able to determine a presence orabsence of a specified blood antigen in a sample of whole blood. Itmight have been expected that cells and other species normally presentin whole blood would interfere with such an assay.

SUMMARY OF THE INVENTION

The present invention provides in a first aspect a method of performinga competitive binding immunoassay for determining in vitro the presenceof a specified blood antigen phenotype in a sample of whole blood, whichcomprises:

mixing the sample of whole blood with a predetermined amount of antibodyspecific to said blood antigen phenotype, so as to bind the antibody toany of said antigen present in the sample of whole blood;

bringing the mixture into contact with a substrate upon which antigen ofsaid phenotype has been immobilized such as to bind any remainingunbound antibody to said immobilized antigen, thereby forming animmobilized antigen-antibody complex;

washing the immobilized antigen-antibody complex and;

estimating the amount of immobilized complex and consequently thespecified blood antigen phenotype.

The present invention in a further aspect provides a competitive bindingimmunoassay kit for determining the presence of a specified bloodantigen phenotype in a sample of whole blood which, comprises;

a substrate having immobilized thereon substantially pure blood antigenof said phenotype.

a supply of antibody for specifically binding to said antigen, and

means for enabling detection of said specifically bound antibody.

The immunoassay of the present invention is described as a competitivebinding immunoassay essentially because any blood antigen of saidspecified phenotype present in the whole blood sample competes with saidimmobilized antigen, for the predetermined amount of antibody.

The immunoassay can be used to detect the presence of any suitableintrinsic blood cell antigen of a specified phenotype (excluding foreignspecies such as viral antigens etc.). The blood antigens includeantigenic determinants on platelet glycoproteins, in particular HPA1antigen of 1 a or 1 b phenotype. Other antigens which are also suitableare glycoproteins or other determinants on blood cells, particularlywhite blood cells (such as granulocytes).

The sample of whole blood may be obtained fresh from an individual to betested, or the blood may be from whole blood stored up to 25 days at 4°C. (for example citrated or EDTA whole blood). The whole blood samplemay be used untreated, or alternatively the whole blood sample can besubjected to a washing step prior to testing (e.g. by diluting theblood, centrifuging it and resuspending in a buffer).

Typically, a sample of serum containing a high titer of antibodyspecific to said blood antigen may be employed in the immunoassay.Alternatively a monoclonal antibody specific to said blood antigen couldbe used.

The substrate upon which said substantially pure antigen of saidphenotype is immobilized, can be any suitable substrate known in theart. Typically this can be paper, plastics such as nitrocellulose, orglass (e.g. microscope slides). Most preferably the substrate is a wellof a plastics microtiter plate, as commonly employed in immunoassays.

The antigen to be immobilized is preferably substantially pure. That is,the antigen is purified away from other blood antigens or bloodcomponents that could affect the immunoassay. It is important that theantigen be substantially pure if the antibody used is in a serum sample.This is to prevent any additional antibodies that may be present in theserum sample, from binding to their respective blood antigen. If amonoclonal antibody is employed, it may not be necessary to highlypurify the antigen, depending on the degree of cross-reactivity of themonoclonal antibody which should be specific to its specified antigen.The antigen used should be purified from a blood source, different tothat being tested. This is to minimize any further antigen-antibodycomplexes which may be formed, from interfering with the immunoassay.

Typically a number of immune (i.e. antigen-antibody) complexes may beformed when bringing the mixture into contact with the substrate, forinstance; a) specified antibody and immobilized antigen, b) specifiedantibody and antigen present in the whole blood, and c) non-specificallybound unspecified antibody to immobilized antigen. The washing serves toremove all complexes except the specified antibody and immobilizedantigen complex.

The mixing of the sample of whole blood with antibody and the bringingthe mixture into contact with the substrate can be carried outconcurrently, or alternatively as two separate steps.

Generally, the substrate bound immune complex is estimated by means ofanti-human IgG or IgM labelled with an appropriate label, such as anenzyme label (e.g. horseradish peroxidase HRP), a radiolabel, afluorescence label or other labelling system known in the art. Using anenzyme label, an enzyme-linked immunosorbent assay (ELISA) may beprovided which is sensitive, specific, cheap, quick and couldpotentially be used for large scale automated screening for whole bloodantigens. Advantageously, it has been found that the incubationsinvolved in the production of the immune complex, and the estimationthereof with labelled antibody can be carried out at room temperatureand so do not require the use of an incubator.

It is envisaged that a particularly preferred immunoassay would involvedetection of HPA1a antigen. The antigenic determinant for HPA1a ispresent on the GPIIIa subunit, so that a competitive binding immunoassayto detect the presence HPA1a antigen in a whole blood sample, wouldinclude immobilized HPA1a antigen. Typically this can be HPA1a typedplatelets (which have been previously genotyped, for instance by usingthe technique described in Williamson, et al (13)), purifiedglycoprotein complex GPIIb/IIIa (prepared, for example, by the processdescribed in Bessos et al (10)), purified glycoprotein GPIIIa, orsynthetically manufactured HPA1a antigen.

In a particularly preferred embodiment of the first aspect, the presentinvention provides a method of performing a competitive bindingimmunoassay for determining in vitro the presence of HPA1a antigen in asample of whole blood, which comprises;

mixing the sample of whole blood with a predetermined amount ofanti-HPA1a antibody, so as to bind the antibody to any HPA1a antigenpresent in the sample of whole blood;

bringing the mixture into contact with a substrate upon which HPA1aantigen has been immobilized, such as to bind remaining anti-HPA1aantibody to the immobilized HPA1a antigen, thereby forming animmobilized HPA1a antigen—anti-HPA1a antibody complex;

washing the immobilized HPA1a antigen—anti-HPA1a antibody complex; and

estimating the amount of immobilized complex and consequently HPA1aantigen.

In a particularly preferred embodiment of the further aspect, thepresent invention provides a competitive binding immunoassay kit fordetermining the presence of HPA1a antigen in a sample of whole bloodwhich comprises;

a multiwell microtiter plate or plates having a well containingimmobilized substantially pure HPA1a antigen;

a supply of anti-HPA1a antibody for specifically binding to said HPA1aantigen, and

means for enabling detection of said specifically bound antibody.

Typically, each well contains 0.05 μg to 0.5 μg of antigen per well;preferably 0.1 μg of antigen per well.

The substrate upon which HPA1a antigen has been immobilized is generallyblocked with a non-specific protein mixture, such as bovine serumalbumin prior to bringing the mixture into contact with the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described by way ofexample only, with reference to the Figures which show;

FIG. 1 shows the distinction between HPA1a positive and negative GPextracts, using anti-serum T.

FIG. 2 shows the distinction between HPA1a positive and negativeplatelets, using anti-serum T and anti-serum C.

FIG. 3 shows the distinction between HPA1a positive and negative samplesusing fresh and stored whole blood.

FIG. 4 shows an assessment of the competitive binding assay performanceusing 33 previously genotyped blood samples, compared to positive andnegative controls.

FIG. 5 shows a representative set of assay results obtained from a studyof 475 blood samples.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1 Purification ofGPIIb/IIIa

The various reagents used were of the Analar grade and were purchasedfrom ICN(UK), Sigma(UK), or Aldrich Chemicals Ltd, (UK). Anti-HPA1a serawere obtained from mothers of babies with NAITP. HRP-conjugated sheepanti-human IgG was provided by the Scottish Antibody Production Unit,and bovine serum albumin (BSA), Boseral, was purchased from OrganonTechnika (Holland). Synthetic peptides and CNBr activated Sepharose 4Bwere purchased from Calbiochem/Novabiochem (UK) and Pharmacia Ltd. (UK)respectively. Genotyped platelets (either separated and washed or inwhole blood) were used to assess the performance of the new CB assay.

Platelet membrane glycoprotein GPIIb/IIIa was extracted and purifiedessentially as described before (10, 11). Briefly, a unit of HPA1a1a,HPA1a1b, or HPA1b1b apheresed platelet concentrate (PC) was washed andextracted with 100 mM octylglucopyranoside and 2 mM phenylmethylsulfonylfluoride in buffer containing 50 mM Tris/HCl and 150 mM NaCl, pH 7.5(TBS). The extract was supplemented with cations and mixed end-over-endwith 2 g of immobilized synthetic peptide GRGDSPK (at 12 mg peptide/gSepharose 4B) overnight at 4° C. The Sepharose beads were thencentrifuged (1,000 for 5 min at room temperature), and the supernatantremoved. The GPIIb/IIIa adsorbed beads were washed several times beforeeluting with cation enriched buffer (1 mM MgCl₂ and 1 mM MnCl₂)containing synthetic peptide GRGDSP. The eluate was dialyzed twice at 4°C. against a liter of Tris/HCl buffer (10 mM Tris/HCl, 150 mM NaCL pH7.4) and stored frozen in aliquots at −40° C. until needed. Typically,about 1 mg of GPIIb/IIIa was removed from each unit of apheresed PC with95% purity.

EXAMPLE 2 Determination of a Suitable Anti-HPA1a Serum and its Abilityto Distinguish Between Soluble GPIIb/IIIa of Different HPA Phenotypes

The binding of three anti-HPA1a sera to coated HPA1a1a was assessed inserial doubling dilutions, of which only two (C and T) exhibitedsatisfactory binding (data not shown) (10). Limiting antibody dilutionsgiving 50% maximum binding (1:40 for both antisera) were used to developthe CB assay. In the first instance, antibody binding in the presence ofserial doubling dilutions of soluble HPA1a1a and HPA1b1b GP (starting at4 ug/ml) was compared. Both antisera (C and T) were found to distinguishbetween the two phenotypes.

EXAMPLE 3 Competitive Binding (CB) Assay

The whole blood or platelet samples obtained from normal volunteers werewashed or diluted in platelet wash buffer consisting of 148 mM NaCl, 5mM glucose, 0.6 mM EDTA, and 20 mM Tris, pH 7.4. The washing ofmicrotiter plate wells in the ELISA was carried out in ELISA wash bufferconsisting of 137 mM NaCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄. 12H₂, 2.7 mMKCl, and 0.05% Tween^(RTM) 20, pH 7.5.

The basic coating, blocking, washing, and color development proceduresof the CB ELISA were as described previously (10,12). Maxisorpimmuno-plates IF (A/S Nunc, Denmark) were coated with 100 μl/well ofpurified HPA1a1a GPIIb/IIIa at 1 μg/ml overnight at 4° C. and blockedwith 5% BSA (in coating buffer) for 1 hr at room temperature (RT) [wellswith coating buffer but without GPIIb/IIIa were included as controls forbackground binding].

Initially, for determining a suitable anti-HPA1a serum, the wells werewashed with ELISA wash buffer and sera added in triplicate in serialdoubling dilutions, at 100 μl/well and incubated for 1 hr. The resultswere plotted on a graph (data not shown) and this resulted in a sigmoidcurve, from which the dilution of antibody giving 50% maximum bindingcould be determined. This “limiting” antibody amount would enablecompetition between cellular HPA1a antigen and the coated antigen forthe antibody.

For development of the CB assay, purified GPIIb/IIIa, citrated or EDTAwhole blood at 1:10 in platelet wash buffer (i.e. 1:20 once mixed withantibody—see below), or washed platelets in platelet wash buffer (all ofvarious phenotypes), were added in triplicate to the wells at 50μl/well. The wells were then supplemented with 50 μl/well of anti-HPA1aserum at 1:20 in ELISA wash buffer (i.e. 1:40 final dilution) and theplate gently shaken (rotatest Shaker, Luckham, UK) for 1 hr at RT. Thisallows competition between coated HPA1a GPIIb/IIIa and its correspondingphenotype on the soluble GP or platelets (either separated or in wholeblood) for the predetermined amount of anti-HPA1a serum. The same HPAphenotype (HPA1a) present in a sample would significantly inhibit thebinding of the antibody to the immobilized antigen in the wells, whereasHPA1b would not. After the incubation, the plates were washed bysquirting ELISA wash buffer 3×100 μl/well up to ten times. Anti-HPA1aantibody bound to the wells was detected by incubation withHRP-conjugated sheep anti-human IgG at 1:400 in ELISA wash buffer (100μl/well), before washing and developing the color as previouslydescribed (12). Specific binding was obtained by subtracting theabsorbence in wells coated with buffer only from those coated withpurified GPIIb/IIIa.

EXAMPLE 4 CB Assay Using GP Extracted from Platelets with Different HPA1Phenotypes

Non-purified extracts obtained from phenotyped washed platelets (byOctyl glucopyranoside detergent extraction as mentioned above) weretested in the CB assay in serial doubling dilutions (starting at 1:10).As shown in FIG. 1 there was a clear distinction between HPA1a1a orHPA1a1b and HPA1b1b GP extracts indicating the suitability of the assayfor determining platelet phenotypes using extracted GP.

EXAMPLE 5 CB Assay Using Washed Platelets from HPA1a and HPA1bVolunteers

Citrated blood samples (10 ml) obtained from an HPA1a and an HPA1bvolunteer were centrifuged at 750 g for 10 minutes, RT, and the buffycoat removed, washed twice with platelet wash buffer, resuspended inwash buffer to similar platelet count, and tested in serial dilutions of10×. HPA1a platelets caused significant inhibition of antibody binding,while HPA1b platelet showed none (FIG. 2). Since antiserum Tconsistently showed a better distinction between the two phenotypes, itwas chosen for routine use in the CB assay. Repeats of this experimentindicated that variation within normal platelet count was not criticalto the observed distinction between the two phenotypes (data not shown).

EXAMPLE 6 CB Assay Using Fresh and Stored Citrated or EDTA Whole BloodObtained from HPA1a and HPA1b Volunteers

Once the applicability of the assay was tested with extracted GP andintact platelets, its applicability to non-manipulated whole blood wasinvestigated. Both citrated and EDTA whole blood was obtained from anHPA1a and an HPA1b volunteer and stored at 4° C., and tested at 1:10 inplatelet wash buffer at various intervals of time. As shown in FIG. 3,the specific absorbence using HPA1a blood was consistently distinct fromHPA1b blood up to day 7 of storage. The procedure described in example 3was followed, with the following exceptions: the wells were washed 7×after incubation with blood on days 1 and 2, and 10× for the rest of thestorage period in order to improve the relatively inferior performanceof the assay on citrated blood on the first two days.

EXAMPLE 7 Assessment of CB Assay Performance by Testing GenotypedSamples Blind

In order to assess the performance of the assay three sets of differenttests were carried out at different times using blind samples obtainedfrom the Cambridge Regional Blood Transfusion Centre. These samplesconsisted of EDTA blood genotyped as described in a recent reviewarticle by Williamson et al. (13). The assay procedure according toexample 3 was followed to assay blood samples at 1:10 dilution usinganti-serum T. In each test HPA1a positive and negative controls storedat 4° C. were included. In the first test the controls were 3 day oldblood samples. In the second test the controls were 8 day old bloodsamples. In the third test the controls were 15 day old samples. The CBassay exhibited a very satisfactory performance (FIG. 4) correctlydetermining the phenotypes of all samples, including 6 HPA1b samples(nos. 1 and 4, first test; nos. 2, 8, 14, second test; and no. 12, thirdtest). Following these experiments, the assay was altered to maximizethe signal to noise ratio by the addition of an initial whole bloodwashing step: 5 ml blood at 1:10 diluted in platelet wash buffer is spunat 750 g for 10 minutes at room temperature, and the cells resuspendedin 1 ml platelet wash buffer (see FIG. 5 for improvement in signal tonoise ratio).

EXAMPLE 8 A Comparative Study of the CB Assay Against the Capture-P KitAssay

The CB assay of the present invention was compared against thecommercially available Capture-P kit assay (Immucor Inc., U.S.A.). Theprocedure according to example 3 was followed together with apreliminary washing step of whole blood using a 1:10 dilution inplatelet wash buffer.

475 samples were tested and the CB assay and Capture-P kit assay were in100% agreement (468 HPA1a positive and 7 HPA1a negative). The meanabsorbances of HPA1a and HPA1b samples were 0.12 (SD 0.08) and 0.49 (SD0.16) respectively, P<0.0005, Student's t test (FIG. 5a). An example ofthe histograms obtained using the CB assay is shown in FIG. 5b, wheresample number 3 is clearly HPA1b. (Positive and negative imply HPA1apositive and HPA1a negative [i.e. HPA1b], controls respectively).Controls in these series of tests comprised HPA1a or HPA1b whole bloodstored for up to 25 days at 4 C. (preliminary results also showed thatsoluble HPA 1a and 1b GP could serve as alternative controls—data notshown).

Although there was background binding in the CB assay (in wells notcoated with antigen), it was possible to obtain a clear distinctionbetween HPA1a positive and negative platelet phenotypes. Generally twofactors appear critical for this distinction: firstly, the use of astrong anti-HPA1 antiserum; and secondly, excessive washing of wells atthe end of incubation with whole blood. The excessive washing wasrequired not only to remove non-specifically bound IgG but also anybound anti-GPIIb/IIIa antibodies which may be present occasionally insome samples (such as those obtained from immune thrombocytopeniapatients). Such antibodies are probably not as strong as the anti-HPA1aantibody and thus may not pose a problem in the assay.

The present assay is ideally suited for large scale screening ofantenatal samples for the following reasons; it only requires the use ofnon-manipulated blood; it incorporates only small amounts of anti-HPA1aserum (2.5 μl/well); and is easily automatable. Large scale screeningshould detect mothers at risk from FAITP or NAITP so that they could befollowed closely during pregnancy to detect signs of fetalthrombocytopenia with or without the emergence of anti-HPA1a antibodies.

REFERENCES

1. Newman P: Platelet GPIIb/IIIa: molecular variations and alloantigens.Thromb. Haemost. 66: 111, 1991.

2. Mueller-Eckhardt C, Mueller-Eckhardt G, Willen-Ohff H, Horz A,Kuenzlen E, O'Neill G J, Schendel D J; Immunogenecity of and immuneresponse to the human platelet antigen ZW^(a) is strongly associatedwith HLA-B8 and DR3. Tissue Antigens 26:71, 1985.

3. Mueller-Eckhardt C: Post-transfusion Purpura. Br. J. Haematol. 64:419, 1986.

4. Gafni A, Blanchette, V S: Screening for neonatal alloimmunethrombocytopenia: an economic perspective. In: Current Studies inHaematology and Blood Transfusion (eds. Decary F, Rock G), 140. Karger,Basel, 1988.

5. Waters A, Murphy M, Hambley H, Nicolaides K; Management of allommunethrombocytopenia in the fetus and neonate. In: Clinical and BasicScience Aspects of Immunohaematology (ed. Nance S J), 155. AmericanAssociation of Blood Banks, Arlignton, Va., 1991.

6. Borne Kr Von Dem D E G, V E R Heught F W A, OosterhofF, Riesz Von E,Brutel de la Riviere A, Engelfriet C P: A simple immunofluorescence testfor the detection of platelet antibodies. Br. J. Haematol. 39; 195,1978.

7. Freedman J, Hornstein A: Simple method for differentiating betweenHLA and platelet specific antibodies by flow cytometry. Am. J. Haematol.38: 314, 1991.

8. Kiefel V: The MAIPA assay and its application in immunohaematology.Transfusion Med. 2. 181, 1992.

9. Metcalfe P, Doughty H A, Murphy M F, Waters H: A simplified methodfor large-scale HPA-1a phenotyping for antenatal screening. TransfusionMed. 4: 21, 1994.

10. Bessos H, Goldschmeding R, Borne Von Dem A K R, Atkinson A, Murphy WG: The development of a simple and quick enzyme-linked immunosorbentassay for anti-HPA1a (PLA1) antibodies. Thromb. Res. 69: 395, 1993.

11. Kirchhofer D, Pierschbacher M D, Ginsberg M H, Plow E F<Ruoslahti E:Platelet membrane glycoprotein IIb/IIIa: a member of a family ofArg-Gly-Asp-specific adhesion receptors. Science 231: 1559, 1990.

12. Bessos H, Murphy W G: A new competitive binding enzyme-linkedimmunosorbent assay for glycocalicin in plasma and platelet concentratesupernatants. Thromb. Res. 59: 497, 1990.

13. Williamson L M, Bruce D, Lubenko A, Chana H J, Ouwehand W H:Molecular biology for platelet allantigen typing. Transfusion Med. 2:255, 1992.

What is claimed is:
 1. A method of performing a competitive bindingimmunoassay for determining in vitro the presence of a specified bloodantigen phenotype in a sample of whole blood, where said blood antigenphenotype comprises platelet blood antigens, and wherein said methodcomprises: (a) contacting a sample of whole blood with a predeterminedamount of antibody specific to said blood antigen phenotype, so as tobind the antibody to any of said antigen present in the sample of wholeblood; (b) contacting the mixture of step (a) with a substrate uponwhich antigen of said phenotype has been immobilized such as to bind anyremaining unbound antibody to said immobilized antigen, thereby formingan immobilized antigen-antibody complex; (c) washing the immobilizedantigen-antibody complex; and (d) detecting the amount of immobilizedantigen-antibody complex, wherein the amount of immobilizedantigen-antibody complex indicates the presence of said blood antigenphenotype.
 2. A method of performing a competitive binding immunoassayaccording to claim 1 wherein the whole blood is washed by dilution,centrifugation and resuspension in a buffer prior to contacting theantibody.
 3. A method of performing a competitive binding immunoassayaccording to claim 1 wherein step (a) comprises contacting said sampleof whole blood with a sample of serum containing said predeterminedamount of antibody specific to said blood antigen phenotype.
 4. A methodof performing a competitive binding immunoassay according to either ofclaims 1 or 2 wherein the antibody is a monoclonal antibody specific tosaid blood antigen.
 5. A method of performing a competitive bindingimmunoassay according to claim 1 wherein the substrate is a well of amicrotiter plate.
 6. A method of performing a competitive bindingimmunoassay according to claim 1 wherein the antigen to be immobilizedis purified and as such is purified away from other blood antigens orblood components.
 7. A method of performing a competitive bindingimmunoassay according to claim 6 wherein the antigen to be immobilizedis purified from a blood source different from the subject being tested.8. A method of performing a competitive binding immunoassay according toclaim 1 wherein steps (a) and (b) are carried out concurrently.
 9. Amethod of performing a competitive binding immunoassay according toclaim 1 wherein steps (a) and (b) are carried out as two separate steps.10. A method of performing a competitive binding immunoassay accordingto claim 1 wherein the immobilized antigen-antibody complex is estimatedby means of an anti-human antibody labelled with an appropriate label,selected from the group consisting of an enzyme label, a radiolabel, anda fluorescence label.
 11. A method of performing a competitive bindingimmunoassay according to claim 1 wherein the method is carried out atroom temperature.
 12. A method of performing a competitive bindingimmunoassay according to claim 1 wherein the immunoassay detects thepresence of HPA1a antigen in a whole blood sample and said immobilizedantigen is HPA1a antigen.
 13. A method of performing a competitivebinding immunoassay according to claim 12 wherein the immobilized HPA1aantigen is selected from the group consisting of HPA1a typed platelets,, purified glycoprotein complex GPIIb/IIIa, purified glycoproteinGPIIIa, and synthetically manufactured HPA1a antigen.
 14. A method ofperforming a competitive binding immunoassay for determining in vitrothe presence of HPA1a antigen in a sample of whole blood, wherein saidmethod comprises: (a) contacting the sample of whole blood with apredetermined amount of anti-HPA1a antibody, so as to bind the antibodyto any HPA1a antigen present in the sample of whole blood; (b)contacting the mixture of step (a) with a substrate upon which HPA1aantigen has been immobilized, such as to bind remaining anti-HPA1aantibody to the immobilized HPA1a antigen, thereby forming animmobilized HPA1a anitgen-anti-HPA1a antibody complex; (c) washing theimmobilized HPA1a antigen-anti-HPA1a antibody complex; and (d) detectingthe amount of immobilized complex, wherein the amount of immobilizedantigen-antibody complex indicates the presence of HPA1a antigen in saidsample of whole blood.
 15. A competitive binding immunoassay kit fordetermining the presence of HPA1a antigen in a sample of whole bloodwhich comprises; at least one microwell microtiter plate having at leastone well containing immobilized purified HPA1 a antigen; anti-HPA1aantibody for specifically binding to said HPA1a antigen, and means fordetecting said specifically bound antibody.
 16. A competitive bindingimmunoassay kit according to claim 15 wherein said well containingimmobilized HPA1a antigen contains 0.05 μg to 0.5 μg of antigen.
 17. acompetitive binding immunoassay kit according to claim 15 wherein thesubstrate upon which HPA1a antigen has been immobilized has been blockedwith a non-specific protein mixture.